Microbiology of Barrier Component Analogues of a Deep Geological Repository by Rachel Beaver A thesis presented to the University of Waterloo in fulfillment of the thesis requirement for the degree of Master of Science in Biology Waterloo, Ontario, Canada, 2020 ©Rachel Beaver 2020 Author’s Declaration This thesis consists of material all of which I authored or co-authored: see Statement of Contributions included in the thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Statement of Contributions Chapter 2 The Tsukinuno bentonite sampling was coordinated by Erik Kremmer (NWMO). The Opalinus core was received from Niels Burzan and Rizlan Bernier-Latmani (École Polytechnique Fédérale de Lausanne, Switzerland). The Northern Ontario crystalline rock core sampling was coordinated by Jeff Binns (Nuclear Waste Management Organization). Sian Ford (McMaster University) swabbed the outer layer of the Northern Ontario crystalline rocK core and crushed the inner layer. Melody Vachon (University of Waterloo) assisted with the cultivation of anaerobic heterotrophs and SRB. iii Abstract Many countries are in the process of designing and implementing long-term storage solutions for used nuclear fuel. Like many of these countries, Canada is considering a deep geological repository (DGR) wherein used fuel bundles will be placed in copper-coated carbon steel used fuel containers encased in bentonite buffer boxes. Previously published research has simulated aspects of a DGR experimentally to identify DGR conditions that would prevent microbial activity. Although such microcosm-type experiments can observe microbial growth and activity over relatively limited time frames, a DGR will remain functional for at least a million years, and will be exposed to fluctuating environmental conditions. To address these temporal limitations, there is value to examining ancient natural barrier components that serve as analogues for future DGR components. My research explored the abundance, viability, and composition of microorganisms in ancient natural DGR barrier component analogues using a combination of cultivation- and DNA-based approaches. Samples were obtained from the Tsukinuno bentonite deposit (Japan) that formed in the late Miocene (~10 million years ago), the Opalinus clay formation (Switzerland) that formed approximately 174 million years ago, and Canadian shield crystalline rock from Northern Ontario. Given sample logistics and site-access considerations, most cultivation and molecular analyses focussed on the Tsukinuno bentonite clay samples, although the other site samples were included for comparison using a subset of techniques. Aerobic heterotroph abundances were relatively low for all Tsukinuno bentonite samples, ranging from 4.1 x 101 to 1.1 x 104 colony forming units per gram dry weight (CFU/gdw). Anaerobic heterotrophs were only detected in half of the Tsukinuno bentonite samples and ranged in abundance from 4.5 x 101 CFU/gdw to 9.9 x 102 CFU/gdw. Sulfate-reducing bacteria were only detected in one sample by cultivation, with a very low estimate of 5 MPN/gdw. Overall, culturable microbial abundances were lower than nucleic acid targets detected by quantitative PCR (Wilcoxon signed ranks test; p <0.05). A range of 3.2 x 101 to 7.2 x 107 (average of 5.7 x 106) 16S ribosomal RNA (16S rRNA) gene copies/gdw was iv detected in the Tsukinuno bentonite samples, and averages of 1.2 x 103 16S rRNA gene copies/gdw and 1.1 x 103 16S rRNA gene copies/gdw were measured for the Opalinus clay and Northern Ontario crystalline rock (NOCR) core samples, respectively. Because this research did not use cultivation-based approaches for Opalinus clay and NOCR samples, and PCR-based technique cannot distinguish DNA from cells and extracellular DNA, future research would be required to determine whether the detected DNA was associated with viable microorganisms. Analysis of 16S rRNA gene amplicons revealed that three of the Tsukinuno bentonite samples were dominated by putative aerobic heterotrophs and fermenting bacteria from the Actinobacteria phylum, whereas five of the Tsukinuno bentonite samples were dominated by sequences similar to those from acidophilic chemolithoautotrophs capable of sulfur reduction. The remaining two Tsukinuno bentonite samples had nucleic acid profiles below reliable detection limits, generating inconsistent replicate 16S rRNA gene profiles and probable contaminant sequences. The NOCR and Opalinus clay samples also yielded inconsistent replicate 16S rRNA gene profiles, implying undetectable nucleic acids in those rock samples and profiles indistinguishable from contaminant sequences. Total genomic DNA from Tsukinuno clay cultures was extracted and 16S rRNA genes were amplified and sequenced. Representatives of the phyla Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes were identified in the aerobic heterotroph cultures, and additional bacteria from the phyla Actinobacteria and Firmicutes were isolated in the cultures of anaerobic heterotrophs and fermenting bacteria. Only one nucleic acid sequence detected from a culture was also detected in its corresponding clay sample, suggesting that nucleic acids from culturable bacteria were relatively rare within the clay samples. Sequencing of DNA extracted from the sulfate reducing bacteria culture revealed that the taxon present in the culture was affiliated with the Desulfosporosinus genus, which was not detected in the corresponding clay sample. v Acknowledgements I would like to thank everyone who helped and supported me throughout the completion of this degree. First, I would like to thank my supervisor, Josh Neufeld, for his continued support and enthusiasm, and for providing the resources necessary to complete this research. I would also like to thank the remainder of my committee, Barb Butler and Trevor Charles, for their opinions and insight. Thank you to the Neufeld lab members for their continued input and for making the lab such an enjoyable place to spend the last two years. I would like to specifically thank Katja Engel for her continued input and help with troubleshooting throughout this project, and Daniel Min and Jackson Tsuji for technical support with QIIME2, R, and Python. Thank you to the people who collected the clay and rock samples, without which this research would not have been possible, and to the Nuclear Waste Management Organization, Ontario Research Fund, and the Natural Sciences and Engineering Research Council for funding this research. Finally, I would like to thank my family and friends for their ongoing support. vi Table of Contents Author's Declaration ................................................................................................................. ii Statement of Contributions ...................................................................................................... iii Abstract .................................................................................................................................... iv Acknowledgements .................................................................................................................. vi List of Figures .......................................................................................................................... ix List of Tables ........................................................................................................................... xi List of Abbreviations .............................................................................................................. xii Chapter 1 Introduction and literature review ............................................................................ 1 1.1 Canadian used nuclear fuel ............................................................................................. 1 1.2 Storage of used nuclear fuel ............................................................................................ 2 1.3 Bentonite clay .................................................................................................................. 3 1.4 Environmental conditions of a DGR ............................................................................... 4 1.5 Importance of studying DGR microbiology .................................................................... 6 1.6 Biogeochemical cycling relevant to a DGR .................................................................... 6 1.6.1 Primary Production in a DGR environment ............................................................. 7 1.6.2 Chemoorganoheterotrophic metabolisms ................................................................. 9 1.6.3 Sulfide sequestration ............................................................................................... 10 1.7 Preventing microbial growth in a DGR ......................................................................... 12 1.8 Methods for characterizing the microbiology of bentonite clay ................................... 13 1.9 Objectives ...................................................................................................................... 14 1.9.1 Sample sites ............................................................................................................ 15 Chapter 2 Materials and Methods ........................................................................................... 16 2.1 Sampling and